PROJECT SUMMARY/ABSTRACT Staphylococcus aureus is a significant cause of morbidity and mortality due to a remarkable capacity to colonize multiple host tissues. Consistent with this, S. aureus is the leading cause of skin and soft tissue infections, bacteremia, osteomyelitis and endocarditis. Treating infections can be exceedingly challenging due to the prevalence of antibiotic resistant isolates, which necessitate the development of new therapeutic strategies. To proliferate within diverse tissues, S. aureus acquires essential nutrients by exploiting abundant nutrient reservoirs present in the host environment. S. aureus nutrient iron acquisition strategies have been studied for decades; however, the mechanisms employed by this pathogen to obtain the equally important nutrient sulfur during infection are not known. Reduced and oxidized forms of the sulfur-containing molecules, glutathione and cysteine, are abundant in host tissues and support in vitro proliferation of S. aureus. Whether these molecules satisfy the sulfur requirement during pathogenesis is unresolved, because we do not understand how S. aureus imports and catabolizes these molecules. To elucidate the mechanisms S. aureus employs to acquire host-derived glutathione, we completed a forward genetic screen and identified mutants that fail to grow in medium supplemented with glutathione as the sole source of sulfur. A reverse genetic approach was pursued to identify potential cysteine transporters. We constructed mutants inactivated for homologues of putative oxidized cysteine transporters and show that the mutated strains are impaired for oxidized cysteine utilization in vitro. Notably, one of the importers provides a competitive advantage in liver colonization in a murine model of systemic infection. These preliminary data represent identification of the first S. aureus sulfur acquisition systems and support the hypothesis that during infection, S. aureus targets abundant host-derived sulfur-containing molecules to satisfy the sulfur requirement. The proposed work will test this hypothesis by (i) establishing the mechanisms that support S. aureus import and catabolism of host-derived GSH, (ii) identifying S. aureus reduced and oxidized cysteine acquisition strategies during infection, and (iii) determining sulfur source abundance and distribution at the host-pathogen interface. Understanding how the host immune response to infection impacts sulfur source availability in tissues is also a goal of this study. The completion of this work will reveal the mechanisms S. aureus employs to acquire host-derived sulfur sources during colonization of distinct tissues. This work will provide novel therapeutic strategies to combat antibiotic resistant S. aureus by impeding nutrient sulfur acquisition. We predict that S. aureus sulfur source acquisition strategies are likely conserved in other bacterial pathogens, broadening the scope and impact of the proposed work.